This invention relates to methods for altering the behavior of the reproductive system of the female mammal. More specifically, the invention relates to methods for controlling fertility using substances which can be isolated from the zoapatle plant.
The name "zoapatle" is applied in Mexico to a group of plants of the family Compositae and tribe Heliantheae; the name principally designates the plant Montanoa tomentosa ssp. tomentosa, but the term is also applied to various other Montanoa species including M. tomentosa ssp. microcephala and xanthiifolia, M. frutescens, M. leucantha ssp. arborescens and leucantha, M. grandiflora, M. speciosa and M. mollissima. To the untrained eye, all these plants are similar in appearance and indeed the material sold as zoapatle leaf often turns out to be a mixture of leaves from different species. Accordingly, the term "zoapatle" will be used hereinafter to cover all of them.
It has been known for centuries that extracts of zoapatle can be used as labor inducers or to increase uterine contractions during labor, as well as to decrease post-partum bleeding. It has also been known since 1866 that such extracts can be used as menses inducers. See, inter alia, Alfaro, R., Cihopatli o Zoapatle, Gaceta Medica De Mexico, 3, 47-48 (1866); del Pozo, E., Aztec Pharmacology, Ann. Rev. Pharm. 6, 9-18 (1966); and Ortiz, de Monte Llano, B., Empirical Aztec Medicine, Science, 188, 215-220 (1975).
As with any pharmaceutical agent of natural origin, a major problem in using zoapatle extract in a controlled, scientific manner is to obtain a product of standard potency. One previous attempt to produce a product of standard potentcy is described in U.S. Pat. No. 4,006,227 granted Feb. 1, 1977 to Gallegos et al. This patent describes a composition derived from M. tomentosa leaves and stems by aqueous extraction with a controlled ratio of vegetable material to water at a controlled temperature. Unfortunately, even these controls are not altogether successful, since, as with many other plants, research indicates that the amount of active ingredient in Montanoa plants varies with the species, and with the conditions under which the plant is grown, including such factors as climate, humidity, soil composition and altitude. Thus, even standardizing the amount of plant material used to prepare an aqueous zoapatle extract will not result in a composition of completely reproducible properties.
Furthermore, like most natural products, the crude aqueous extract of zoapatle is known to be a very complex mixture. At least nine different compounds have been isolated from zoapatle extract and hitherto it has not been possible to identify which of these nine products is responsible for the characteristic action of the crude extract. The nine compounds involved are montanol, zoapatanol, tomentosin, montafrusin, monoginol, (-)-kaura-9(11),16-dien-19-oic acid (hereinafter sometimes referred to simply as kauradienoic acid), kaurenoic acid, zoapatlin and monoginoic acid. The structures and extraction of these compounds are described in the following papers:
Levine, S. D. et al, Zoapatanol and montanol, novel oxepane diterpenoids from the Mexican plant zoapatle (Montanoa tomentosa), J. Am. Chem. Soc., 101, 3404-5 (1979); PA1 Geissman, TA, Griffin, TS, Sesquiterpene lactones, tomentosin from Montanoma tomentosa (cerv.), Rev. Lat. Quim., 2, 81-83 (1971); PA1 Quijano, L. et al, Montafrusin, a new germacrolide from Montanoa frutescens, Phytochemistry, 18, 843-45 (1979); and PA1 Caballero, Y., Walls, F., Productos naturales del zoapatle, Bol. Inst. Quim., UNAM, 22, 79-102 (1970).
The structures of kaurenoic acid, (-)-kaura-9(11),16-dien-19-oic acid, monoginoic acid, zoapatanol and montanol are also shown in FIG. 5 of the accompanying drawings. It is of course entirely possible that there are other pharmacologically active constituents of the zoapatle aqueous extract which have not yet been isolated.
For obvious reasons, faced with such a complex mixture, research workers have attempted to separate the pure compounds and to determine which one or more of them is responsible for the characteristic pharmaceutical properties of the crude extract. For example, U.S. Pat. Nos. 3,996,132, granted Dec. 7, 1976, 3,986,952, granted Oct. 19, 1976, 4,060,604, granted November 1977, 4,112,079, granted September 1978, and 4,127,651, granted November 1978, all of which are assigned to Ortho Pharmaceutical Corporation, Raritan, N.J. 08869, describe extraction and chromatographic techniques by which two pharmaceutically active compounds, now known to be the oxepane diterpenoids zoapatanol and montanol, can be extracted from the crude aqueous extract of zoapatle. In addition, the total synthesis of (.+-.)-zoapatanol has been accomplished, as described in Nicolaou, K. C., Claremon, D. A., Barnett, W. T., Total Synthesis of (.+-.)-zoapatanol, J. Am. Chem. Soc., 102, 6611-12 (1980). However, further research apparently indicates that neither zoapatanol nor montanol has a pharmaceutical activity corresponding to that of the crude zoapatle extract itself. For example, the aforementioned Nicolaou et al. paper states that (.+-.)-zoapatanol in vitro shows constriction of the cat coronary artery and contraction of isolated guinea-pig ileum, but fails to contract the non-pregnant rabbit and rat uterus, whereas the crude zoapatle extract elicits a clear a reproducible response upon the uterine contractility of the non-pregnant rabbit and vascular relaxation, without altering intestinal peristalsism, in rabbits, guinea-pigs, monkeys and humans.
Not only has the search for the "active principle" of crude zoapatle extract been hindered by the complexity of the mixtures and the difficulty of isolation of pure compounds therefrom, it has also been bedevilled by the lack of consistency in the results achieved with varying animal models. Because of the length, cost, and complexity of in vivo evaluation of zoapatle-derived compounds, it is necessary to test biological activity in vitro. Unfortunately, hitherto there has been no systematic attempt to investigate the appropriateness of various animal models and their correlation with the known effects in primates. As a result, the literature is filled with a great number of biological observations showing apparently contradictory or conflicting results. For example, the aforementioned Caballero et al. paper reports that monoginoic and kaurenoic acids isolated from zoapatle roots showed a complete lack of in vitro activity. These results were based upon experiments with isolated rat uterine strips and observations that these two acids failed to increase the tone or frequency of the contractions of such isolated rat uterine strips. It is now known that rat uterine strips do not increase tone or frequency of contractions when exposed to crude zoapatle extract and in fact display the opposite effect of relaxation. On the other hand, we have found that the effect of zoapatle-derived compounds on isolated strips of guinea pig uterus does correlate correctly with the activity of such compounds in female mammals.
There is thus a need for identification of the active principle of zoapatle. We have now identified this active principle.